内毒素上调肝细胞表达CD14基因和蛋白的实验研究

Lipopolysaccharide upregulater expression of CD14 gene and CD14 proteins of hepatocyter in rats

目的 观察内毒素血症时肝组织和游离肝细胞中ＣＤ１４基因和ＣＤ１４蛋白的表达。方法 经尾静脉注入脂多糖（ＬＰＳ， Ｅ ｃｏｌｉ Ｏ１１１．Ｂ４）建立大鼠急性内毒素血症动物模型，并用原位胶原酶灌注法分离肝细胞。用流式细胞仪（ＦＣＭ）测定异硫氢酸荧光素（ＦＩＴＣ）－ＣＤ１４阳性肝细胞数及其荧光强度。同时用逆转录－ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ法测定肝组织和肝细胞中ＣＤ１４ｍＲＮＡ和ＣＤ１４蛋白的表达。结果 ＦＣＭ显示：内毒素血症大鼠６ｈ和１２ｈＦＩＴＣ－ＣＤ１４阳性细胞数明显增多，荧光强度也明显增加。ＲＴ－ＰＣＲ显示，肝组织和肝细胞中ＣＤ１４ｍＲＮＡ的表达在３ｈ时明显增强，６ｈ达高峰，２４ｈ恢复正常水平；Ｗｅｓｔｅｒｎ ｂｌｏｔ分析示肝组织和肝细胞中ＣＤ１４蛋白的表达在６ｈ明显增高，１２ｈ达高峰，２４ｈ仍有一定表达，各时相点间比较有显著差别（Ｐ＜０．０１）。结论 内毒素血症时能明显上调肝细胞ＣＤ１４基因和ＣＤ１４蛋白的表达。

Objective: To observe the expression of CD14 mRNA and its protein in rat hepatocytes during endotoxemia. Methods The acute endotoxemia model of wistar rats was established by injection of a dose of LPS (5 mg/ kg, Escherichia colt O111:B4) via the tail vein, then the rats were sacrificed at 3, 6, 12, and 24 hour, respectively. Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique. Hepatocytes and hepatic tissue were collected to measure the expression of CD14 mRNA and CD14 protein by reverse transcriptase-polymerase chain reaction (RT-PCR) or western blot analysis. The binding of fluorescein isotheocyanate (FITC)-CD14 polyclone antibody to isolated hepatocytes was also assessed by flow cytometric analysis (FCM). Results In the rats with endotoxemia, the expression of CD14 mRNA and CD14 protein in hepatic tissue and isolated hepatocytes were stronger at 6, 12 and 24 hour than that in control rats (p<0.01). FCM showed that positive cell numbers of FITC-CD14 in the rats with endotoxemia increased when compared with normal control group (p<0.01). Conclusions LPS can markedly upregulate the expression of CD14 mRNA and CD14 proteins in hepatic tissue and isolated hepatocytes.